NIRF/PET imaging of γδ T cells via metabolic glycoengineering and bioorthogonal labeling

Overview of γδ T cell labeling and NIRF/PET imaging strategy via metabolic glycoengineering and bioorthogonal click chemistry. [Display omitted] •Bioorthogonal labeling strategy enables NIRF/PET imaging to visualize γδ T cells.•Cy5.5-γδ T cells showed strong NIRF signal, allowing NIRF imaging up to 10 days.•Pre-targeting strategy enables the PET imaging of γδ T cells with short half-life radionuclides.•Dead-γδ T cells also shows high tumor targeting and good tumor retention, rendering it a high potential drug delivery system. Non-invasive tracking of γδ T cells may help to predict patient responsiveness and therapeutic efficacy. In this study, we developed an efficient γδ T cell NIRF/PET imaging strategy by combining metabolic glycoengineering and bioorthogonal click chemistry. Initially, γδ T cells were incubated with N-azidoacetyl-D-mannosamine-tetraacylated, and azide (N3) groups were then incorporated onto the surface of γδ T cells via metabolic glycoengineering to obtain N3-γδ T cells. Next, Cy5.5 dibenzylcyclooctyne (DBCO-Cy5.5) and the short half-life 68Ga-labeled chelator 68Ga-NETA-DBCO were conjugated with N3-γδ T cells via in vitro or in vivo bioorthogonal metal-free click chemistry for NIRF and PET imaging, respectively. In Daudi tumor-bearing mice, Cy5.5-labeled γδ T cells migrated specifically to tumor sites, and adoptive cells were observed clearly via non-invasive NIRF imaging over 10 days. Furthermore, in the pre-targeted PET images, high tumor uptake of 68Ga-NETA-DBCO was observed in mice three days post-injection of N3-γδ T cells, which was 1.5-fold higher than that of control mice. Interestingly, dead-Cy5.5-γδ T cells also showed high tumor targeting and good tumor retention, peaking at day 1 and retaining NIRF signal up to 10 days, indicating a high potential of γδ T cells as a drug delivery system. These findings demonstrated that a bioorthogonal labeling method followed by NIRF/PET imaging maps the biodistribution, migration, and tumor-homing efficiency of adoptive γδ T cells in vivo, representing a new potential γδ T cell imaging technology.